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PCT

WORLD INTELLECTUAL PROPERTY ORGANIZATION
International Bureau

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(51) International Patent Classification 6 :
G01N 27/42, 33/487

(11) International Publication Number: WO 97/08544

(43) International Publication Date: 6 March 1997 (06.03.97)

(21) International Application Number: PCT/US96Y13844

(22) International Filing Date: 21 August 1996 (21.08.96)

(30) Priority Data:

60/002,636

22 August 1995 (22.08.95) US

(71) Applicant: ANDCARE, INC. [US/US]; Suite 152, 2810

Meridian Parkway, Durham, NC 27713 (US).

(72) Inventors: WGJCIECHOWSKI, Marek; 102 Otterbein Court,

Cary, NC 27513 (US). EBELING, Frederick, A.; 106
Larkspur Lane, Cary, NC 27513 (US). HENKENS, Robert,
W4 2116 Pershing Street, Durham, NC 27705 (US).
NASER, Najih, A.; 7834 Massey Chapel Road, Durham,
NC 27713 (US). O'DALY, John, P.; 112 Jasmine Court,
Carrboro, NC 27510 (US). WEGNER, Steven, E.; 204
Donegal Drive, Chapel Hill, NC 27514 (US).

(74) Agent: KJTCHELL, Barbara, S^ Arnold, White & Durkee,
P.O. Box 4433, Houston, TX 77210 (US).

(81) Designated States: AL, AM, AT, AU t AZ, BB, BG, BR, BY,
CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, HU,
IL, IS, JP, KE, KG, KP, KR, KZ, LK, LR, LS, LT, LU,
LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO,
RU, SD, SE, SG, SI, SK, TJ, TM, TR, TT, UA, UG, UZ,
VN, ARIPO patent (KE, LS, MW, SD, SZ, UG), Eurasian
patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European
patent (AT, BE, CH, DE, DK, ES, H, FR, GB, GR, IE, IT,
LU, MC, NL, PT, SE), OAPI patent (BF, BJ, CF, CG, CI,
CM, GA, GN, ML, MR, NE, SN, TD, TG).

Published

With international search report

Before the expiration of the time limit for amending the
claims and to be republished in the event of the receipt of
amendments.

(54) Title: HANDHELD ELECTROMONTJDR DEVICE

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(57) Abstract

An apparatus is disclosed which is a microprocessor based instrument designed to conveniently and rapidly measure various analytes
in environmental and biological samples. The instrument operates as a stand-alone unit powered by a battery or a DC power module
and may be equipped with a communication port allowing uploading test results to a computer. Several unique electronic, microchip and
software configurations were developed for the device to make it a portable, low-cost, safe, automated and simple-to-operate instrument
particularly adapted for precise and accurate measurement of metal ions such as heavy metals such as lead in human blood.

FOR THE PURPOSES OF INFORMATION ONLY

Codes used to identify States party to the PCT on the front pages of pamphlets publishing international
applications under Ihe PCT.

United Kingdom

Malawi

Austria

Georgia

Mexico

Australia

Guinea

Niger

1H.il. «.tn.i ll»

ncuicranos

Barbados

Greece

Belgium

Hungary

Norway

Burkina Faso

Ireland

New Zealand

Bulgaria

Italy

Poland

Japan

Portugal

Brazil

Kenya

Romania

Belarus ,

Kyrgystan

Russian Federation

Canada

Democratic People's Republic

Central African Republic

of Korea

Sweden

Congo

Republic of Korea

Singapore

Switzerland

Kazakhstan

Slovenia

Coted'fvoire

Slovakia

Cameroon

Sri Lanka

Senegal
Swaziland

China

Liberia

Czechoslovakia

Lithuania

Chad

Czech Republic

Luxembourg

Togo

Germany

Latvia

Tapkistan

Denmark

Monaco

Trinidad and Tobago

MJ>

Republic of Moldova

Ukraine

Spain

Madagascar

Uganda

Finland

Mali

United States of America

France

Mongolia

Uzbekistan

Gabon

Mauritania

Viet Nam

WO 97/08544

PCT/US96/13844

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DESCRIPTION

HANDHELD ELECTROMONITOR DEVICE

5 BACKGROUND OF THE INVENTION

A portion of this patent document contains material which is subject to copyright
protectioa The copyright owner has no objection to the facsimile reproduction by any one
of the patent disclosure, as it appears in the Patent and Trademark Office patent files or
10 records, but otherwise reserves all rights whatsoever.

Fidd of the Invention

The invention relates generally to a convenient microprocessor based instrument
adapted to measure low levels of analytes in fluids. More particularly, the disclosed devices
15 are automated, portable electrochemical instruments designed to accept various chemically
modified electrode sensors and to rapidly and accurately measure low levels of various
analytes. Hand-held dectromonitors are described that are capable of employing various
electrochemical analytical techniques for the precise and accurate measurement of multiple
analytes in a wide range of fluids.

Description of the Related Art

Recently, the development of highly efficient electrochemical stripping sensors
based on colloidal gold has resulted in the development of rapid, simple tests for
determining trace amounts of contaminants, particularly heavy metals and especially

25 undesirable en v ironmental toxins such as lead and mercury. The sensors for detection of
these metals are basically colloidal gold modified electrodes where the surface appears to ,
act as a microdectrode array, possibly providing a rationale for the superiority of these
electrodes over bulk gold electrodes. The preparation of colloidal gold electrodes is
described in several patents, including U.S. Patent Nos. 5,334,296; 5,391,272; 5,217,594;

30 and 5,368,707 all of which are incorporated herein by reference and in their entirety.

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PCT/US96/13844

CoDoidal gold based electrodes have been used not only for potentiometric
measurement of analytes, but also in square wave coulometric determination of metal ion
levels. Square wave oculometry (SWQ combines fast scanning square wave vokammetry
5 with coulometric measurement of the signal and has been used in some applications as
preferable to differential pulse vohammetry. The advantages of this method with respect to
sensitivity and speed over other voltammetric techniques is discussed in detail in U.S.
Patent No. 5,468,366, the entire disclosure of winch is herein incorporated by reference.
The reference particularly mentions that one advantage of SWC analysis for measurements
10 involving microelectrode arrays is that it does not require removal of dissolved oxygen
from the sample solution in contrast to other stripping techniques.

SUMMARY OF THE INVENTION

15 In a general aspect the invention employs combinations of electrodes in an

electrolyte to generate electrical signals which are indicative of the concentration of an
analyte in the electrolyte. The signals are digitized and processed in digital form to
determine and display the signals. The electrodes in contact with a sample of the electrolyte
are contained in a small fixture or probe which is electrically coupled to a data processing

20 system. This system is housed in a container or housing which is small enough to be hand-
held.

The invention also employs special means for calibrating the instrumentation. One
such means makes use of calibration strips which are coupled to the data processing system
25 in a manner similar to the electrodes. Calibration data from a calibration strip may be
transferred into the system or it may indicate to the system which set of calibration data to
employ from sets which are already stored in the system as in a "lookup" table. In general,
a particular calibration strip is provided for a given set of electrodes, or for a given lot of
such electrodes.

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PCT/US96/13844

An alternate calibration system may employ a microchip on which calibration data
for a given set of electrodes has been stored. A microchip reader on the instrument reads
and transfers the calibration data from the microchip into the data processing system

5 In general, the invention measures a parameter of the electrode signal, notably the

current, for the analysis made by the invention Thus, the system may measure amperage,
or a riwdulated amperage signal to p^nn analyses bas^ on amperometiy, vbhammetry,
square wave coulometry, etc. Anodic stripping vohammetry is particularly preferred in
analyzing for metals in one typical applicatioa

The advent of the colloidal gold electrodes marks a great step forward in the
analysis of metals and contaminants. The present invention marks a further advance in
recognizing the need for an instrument for making such analyses which is readily portable
but also accurate and flexible. Especially attractive is an instalment whkfc is self-contained
15 and sufficiently compact to be hand held. Such an instrument is particularly advantageous
in remote operations and provides results which are not only accurate but also prompt and
cost-saving. It is apparent that such an instrument may be used in the laboratory as weD as
in the field

20 The present invention provides an instrument which combines unique electronic,

microchip and software configurations in a device that is portable, safe, automated and
simple to operate in determining anaryte concentrations in fluid samples. In particular
aspects, an apparatus for analyzing for a selected anaryte in blood, urine or water is
provided. Such a device is small enough to be hand held and can be set to determine

25 virtually any metal ion, in addition to peroxides, glucose, proteins, drugs and pesticides.
The disclosed device may be conveniently set up for use with various electrochemical
analytical techniques such as square wave coulometry, anodic stripping vohammetry, and
amperometry, thereby providing several advantages over other conventional
electrochemical instruments.

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The disclosed microprocessor-based device is designed to perform various tasks
associated with the measurement of electrochemical sensor response. The sensor is used as
a disposable insert with this monitor. In one embodiment, designated the LeadCare™
Monitor (AndCare, Inc., Durham, N.C.)for specifically measuring blood lead levels, the
5 instrument has one mode of operation which is a blood lead level (ELL) measurement
initiated by pressing a push-button switch ("START"), after insertion of the sensor into the
monitor and placing a sample on the sensor. In less than 2 minutes a BLL will be displayed
on the LCD.

10 The monitor incorporates several distinctive functions and features, some of which

are new in this type of device, that include:

Single push-button operation

This is an improvement over portable electrochemical devices available on the
IS market, none of which is for blood lead. They all require at least a few step long set
up/initiation procedures.

Sensor recognition test

A novel feature of the device is that it is set up to ran a test to distingui sh whether a
20 calibration strip or a test sensor is connected when the test sequence is triggered by the
START buttoa

The test is based on die difference in the current vs. time characteristics of a
calibration strip and a test sensor. When a voltage pulse is applied a constant (/.&, time
25 independent) current is generated by resistors of the calibration strip. The test sensor
containing sample solution on the other hand produces current that sharply decays in time.

The device applies a small voltage pulse to the connector and current is sampled
several times over a few millisecond period. If a constant (± 10%) current is detected, the
system assumes that a calibration strip is connected and the software initiates resistance

WO 97/08544 PCT/US96/13844

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measurement of the calibration strip. If a decaying current is detected, the software goes to
the BLL measurement cycle.

Automatic calibration

5 An additional novel feature of the device is the sensor calibration scheme which

eliminates complicated and time consuming manual calibration procedures required by
currently available devices. The scheme involves a resistor network-based calibration strip
and sensor calibration database stored in the EEPROM. It does not require operator
intervention except insertion of the calibration strip and pressing the START button. The
10 system first recognizes that a calibration strip is connected and measures the resistance of
two resistors on the strip. Based on the values obtained, the software activates one of a
plurality of calibration data sets stored in EEPROM to be used for measurements involving
the sensors. Eighty-eight such sets have actually been employed.

IS Calibration strips are plastic slides consisting of printed connecting tracks and

resistive bands whose resistance is laser trimmed to a desired value. A calibration protocol
is carried out on each new batch of sensors to determine which of the calibration data sets
pre-stored in the memory best represents the performance of this lot in the analyte test.
Each of these calibration data sets has its own calibration strip with pre-assigried resistance

20 values.

For example, calibration strip 3F may activate column #3 and offset #6 in the
calibration database. Each manufactured batch of sensors has an appropriate calibration (or
sensor code) strip included to be used for setting up the calibration by the operator.

By inserting the calibration strip into the sensor connector arid pressing the START
button, the operator confirms that the monitor and the sensors within the lot package
function together within the specified measurement bounds of the System. The calibration
strips may be reused at any time during the usable life of the lot of sensors in the package.

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Self diagnostics

A sequence of self diagnostic checks is automatically performed each time the
device is turned oa The self tests are described in the INIT.SRC section. If any of these
tests fails the "ERR" message is displayed on the LCD display and the system is halted, ie. f
5 the device cannot be operated.

Sensor connection and sample solution placement test

A further novel feature of the device is the incorporation of a test to determine
whether the sensor, or calibration strip, is properly connected to the electronics via the
10 connector If a sensor is detected (see Sensor Recognition Test) the sensor connection is
monitored continuously (at one second intervals) during the test sequence. In the event
that an improper sensor connection is detected, an instruction "CHECK SENSOR" is
displayed on the Monitor's LCD display.

IS The same routine tests whether all sensor electrodes are sufficiently covered by

solution of the tested sample. If no resistance due to the sample solution is detected
between the electrodes, a "CHECK SENSOR" message is displayed.

Internal "dummy sensor" test

20 This test is performed after the Monitor is turned oa It checks the AID and D/A

voltage control and other current measuring components of the electronics by running the
scan step of the test sequence after connecting an internal resistor network ("dummy
sensor") to the electronics. The voltage is scanned between selected voltages and the
currents are measured, stored and compared by the software with expected values. Actual

25 voltage scans have included scans between -500mV and -2mV. If test Ms, a system error
message is displayed on the LCD display. This test confirms acceptable performance of
virtually all hardware and software components of the system except the connector which is
checked in a separate self test.

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PCT/US96/13844

LCD display functions

LCD message selection includes:

♦instroctions such as "CHECK SENSOR" and "CALIBRATE"

* warnings such as a battery icon displayed when a low battery status is detected,
5 "ERR: for system error, etc

* test status displays including "SELF TEST," "READY", and "TEST."

Beeper Junctions ~

The Monitor system supports a beeper which provides an additional way of
1 0 signaling to the operator that, for example, a sensor is connected improperly or that the test
is completed.

RS-232 interface

Computer and printer communication functions have been implemented via a buih-
15 in RS-232 interface and fully supported by the software. The interface allows direct
sending of test results to a printer for a hardcopy printout of test. Also, with the use of a
PC computer program, the operator can download new test parameters to the research
version of the device and upload the measured current data and results.

20 In production versions, the device supports an RS-232 protocol in a read only

format that permits external transfers of selected data from the device.

Software functions for improvihgS/N

The signal to noise (S/N) characteristics of the measurement may be improved by:
25 *agnalavOTging;fi>urmeasureinents;

* digital filtration of the forward and reverse currents;

* digital filtration of the difference current obtained by subtraction of filtered
forward and reverse currents;

* baseline subtraction before peak measurement (see paragraph below), and
30 * integration of the peak signal

WO 97/08544 PCT/US96/13844

Baseline subtraction routine

Yet another novel and important feature of the software associated with the
disclosed device is the significant simplification of measurement of peak-shaped
S electrochemical signals. This allows fUU automation of the data treatment process.

The analysis routine works by detecting two minima, one on each side of the peak,
drawing (i.e. calculating) a line through the minima, and then subtracting that line from the
curve to remove the baseline offset

The two minima are chosen by limiting the range that the software searches. Alow
range is defined on the left side of the peak and a high range on the right. Within this range,
minima are found and used to calculate the baseline. The routine works best when repeated
to further improve results. The software performs the analysis in four steps:

a) Find the low point in the two ranges, before and after the peak;

b) calculate the slope of the line drawn between these two points;

c) subtract this baseline's value from the data;

d) repeat 1 through 3 above to improve accuracy.

Battery or AC powermocfule operation

The monitor preferably operates as a stand-alone unit powered by a battery or a
DC power module. The system recognizes whether a battery or an AC power module
powers its electronics. When both AC module and the battery are connected, the system
25 disconnects hsdf from the battery to prolong the battels fife time.

Battery saving and IJCDdisplcyburnort

After 10 minutes without activity when battery powered or after 1 hour when AC
power module is used, the device functions are turned off to save battery and prevent
30 burnout of the LCD display.

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BRIEF DESCRIPTION OF THE DRAWINGS

So that the manner in which the herein described advantages and features of the
5 present invention, as weD as others which will become apparent, are attained and can be
understood in detail, more particular description of the invention summarized above may be
had by reference to the embodiment thereof which is illustrated in the appended drawings,
which drawings form a part of this specification.

10 FIG. 1 shows an example of the potential waveform and the current sample scheme

used with the invention for anodic stripping measurement. This example is specific to
signals for lead in add treated blood using colloidal gold sensors. "F" and "R" represent
the sampling points of forward and reverse currents respectively.

15 FIG. 2 is a graphic representation of a baseline subtraction procedure used to

process a raw electrochemical response into a form used to calculate analyte concentration.

FIG. 3 is a flowchart of various hardware component blocks that comprise a device
of the invention.

FIG. 4 is a basic firmware protocol for the execution of an entire electrochemical
measurement in accordance with the invention.

FIG. 5 is a flowchart of a main loop software framework module which directs the
25 invention to poll and wait for signals to perform a certain activity.

FIGS. 6A-6D is a flowchart of a process control routine which controls the
hardware that is connected to the sensor during a test. The sensor test involves applying a
voltage to a sensor for a specified period of time.

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HG. 7 is a flowchart of a scanning routine which provides the actual data
measuring software for the sensor electrochemistry. This routine scans the voltage from a
first voltage value to a final voltage value.

5 FIG. 8 is a schematic diagram of an exemplary microprocessor system. The

exemplary diagrammed system is built around a MOTOROLA MC 6805 mam
microprocessor which is 8 bit with 176 bytes of internal RAM, 8K bytes of program
memory space, 24 I/O lines, 2 serial interfaces, and a hardware timer.

10 FIG. 9 is a schematic diagram of an exemplary analog circuit which includes a

digital-to-analog (D/A) converter to generate a known voltage and an analog-to-digital
(A/D) converter to measure the current.

FIG. 10 is a schematic diagram of an exemplary display/memory system which
15 stores parameters for the measurement process and data collected while processing the
sample.

FIG. 11 is a schematic diagram of an exemplary power supply which typically
supplies power either by internal batteries or 120V AC power.

FIG. 12 illustrates a version of an exemplary hand-held electronic monitor
indicating the slot where the electrode strip is inserted and where a calibration unit for the
manufactured electrode strip may be inserted to connect into the circuit controlled by the
firmware. Abattery compartment, alternate AC power supply connection, and connector
25 for optional mating with externally supplied calibration programs are indicated.

FIG. 13 is a square wave voltammetric curve of acetaminophen obtained with the
disclosed device and a carbon sensor. Monitor parameters: square wave voftammetry with
100 mV initial potential, 50 Hz frequency, 25 mV amplitude and 2 mV step.

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FIG. 14 is a calibration curve for lead in water using anodic stripping signals
measured by the monitor with a colloidal gold sensor. Monitor parameters: 90s deposition
at -0.5V and stripping by square wave vohammetry at 100 Hz frequency, 25 mV amplitude
and 2 mV steps.

FIG. 15 shows an anodic stripping curve obtained for a mixture of four metals in
0.1M acetate buffer pH 4.2 using the monitor and a carbon sensor with in situ deposited
mercury film Monitor parameters: 240s deposition at -1 4V and stripping by square wave
vohammetry at 1 15 Hz frequency, 25 mV amplitude and 3 mV steps.

FIG. 16 shows amperometric measurement of hydrogen peroxide in 50 mM MES
buffer pH 6.4 using colloidal gold-HRP (horseradish peroxidase) sensors and the disclosed
device. Parameters: -100 mV potential, 4 Hz current sampling rate.

15 FIGS. 17A-17B compare anodic stripping curves as acquired and after processing

by the inventioa The curves are for a sample of 42 \ig per decaliter of lead in add treated
blood using a colloidal gold electrode. Operating parameters included a 90s deposition at -
0.5V and stripping by square wave vohammetry at 80 Hz frequency, 25 mV amplitude and
2 mV steps.

FIG. 18 shows the effect of humidity on the temperature of a sample solution
placed on a colloidal gold sensor. The sample is a 50 nl drop of add treated blood

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention relates to an inexpensive, easy to use and portable electronic
apparatus for tests employing disposable electro sensors. The microprocessor based device
performs various tasks associated with the measurement of current responses generated by
metal ions and non-metal anarytes on different kinds of disposable sensors and electrodes.
30 It can be also used to cany out electrochemical measurements using conventional, reusable

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electrodes and electrochemical cells. The electrochemical functions and techniques
available include:

electrochemical preconditioning of the working electrode by potential steps
5 (pulses),

amperometric measurements at a constant potential applied to the working
electrode,

square wave anodic stripping voltammetry, and
square wave voltammetry.

The amperometric mode may be used for measurements involving immunosensors,
DNA probes and other enzyme based sensors such as colloidal gold sensors for hydrogen
peroxide, atrazine, fertility hormones, cholesterol and others. The square wave anodic
stripping voltammetry has been used for measurement of heavy metals in biological fluids

15 and environmental samples. For example, operation in this mode has been successfully
used in the LeadCare™ Test for detection of blood lead using colloidal gold sensors
(AndCare, Inc., Durham, NC). The same mode of operation can be used for measurement
of lead, cadmium, copper, zinc and other metals in waters and other environmental samples.
The technique of square wave voltammetry can be used to measure anarytes that do not

20 require deposition (preconcentration) on the surface of working electrode. For example,
the device can be operated in the square wave voltaxnmetric mode for measurements of
acetaminophen in aqueous samples.

The device is a three-electrode potentiostat employing an auto current gain
25 switching function which allows a much more rigorous control of the potential applied to
the working electrode during the entire measurement. This feature improves the S/N
characteristics of the device. The device can operate powered by a 9V battery or a DC
power module.

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PCT/US96/13844

The device is a versatile, yet simple to use instrument. It can be used as a
stand-alone for conducting repetitive tests involving the same type of sensors and one set of
operating parameters. In this mode all the parameters are preset either by the device
manufacturer or by means of a calibration/setup microchip "button," and the monitor has
5 only one mode of operation. Similar electrochemical devices available on the market
require complicated setup procedures. The measurement is initiated when the operator
presses START key after inserting the sensor in the sensor connector and placing a drop of
tested sample on the sensor. At the end of measurement the test result is displayed on the
LCD display. This can be regarded as a "black box" mode of operation since the user does
10 not have to be familiar with the operation of the device or with the electrochemistry
involved in the measurement process.

The same device, can also be used as a flexible instrument for development of
electrochemical tests and for other applications involving disposable sensors or other

15 electrodes. This type of operation is designed for more experienced users and requires a
control program and a computer connection. The program provides fiill control of the
functions including changing the type of measurement (araperometric, square wave
voltammetric, eta) and/or including the operating parameters. The measurement data can
be uploaded from the device to the program for display, analysis and storage in the

20 computer memory.

A sequence of self diagnostic checks is automatically performed each time the
device is turned on. If any errors are detected in the hardware or software, an error
message warns the user. Also, the battery status is checked continuously and a battery low
25 icon is displayed if the battery voltage drops below 6.8 V a selected voltage - for example,
6.8V with a 9V battery. When the battery voltage is 6.4 V or less, the device will shut
down aO its functions. Sensor connection and sample drop placement are tested
automatically at the beginning and during the test. In case the sensor is not connected
correctly, or the sample does not cover completely the electrodes on the sensor, or the

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sensor accidentally is disconnected during the test, "CHECK SENSOR" is displayed on the
LCD display and the measurement sequence is aborted.

A temperature correction function may be added to correct for the temperature
S dependence of the entire test process, including the diffusion of dectroactive species at the
electrode surface. It is based on a thermistor probe mounted on the circuit board and
controlled by the microprocessor unit (MPU). The temperature is measured before and
after the test and the test result is extrapolated to the temperature of 25 degrees Celsius
using temperature correction database stored in the EEPROM This function eliminates
10 errors due to variations of the temperature of tested samples.

Sensor preconditioning function involves a set of four independently controlled
steps (pulses) that can be used for electrochemical preconditioning of the sensor. Each
preconditioned step can be set for 1 to 600-second duration and the applied potential from
15 the -2000 to +2000 mV range. These steps may be used without potential applied to the
sensor to aid the operator in controlling the time of other steps in the test procedure.
Potentials applied may be changed gradually between the preconditioning and measurement
steps which very often helps reduce the charging stress on the electrode surface.

20 The sensor housing is a novel device designed to stabilize the temperature of the

sample solution on the sensors and thus reduce the effect of solvent evaporation on the
current signals measured using sensors. This add-on module attaches to the sensor
connector. The housing consists of a plastic part and an aluminum plate forming its
bottom. The slot into which the sensor is inserted forces the bottom surface of the sensor

25 to sHde over the aluminum plate and lay down firmly on its surface. The aluminum plate
functions as a heat sink preventing the cooling of the sensor as the water evaporates from
the tested sohitioa The evaporation effect is particularly significant for the measurements
conducted in dry environments, e.g. , at relative humidities below 40%. The sensor housing
also creates a draft screen for the tested sample which substantially reduces the effect of

30 evaporative cooling caused by draft. The sensor is first inserted halfway into the housing

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so that the electrode area on the sensor is above and ova* the aluminum plate. The tested
sample solution is deposited and spread over the sensor electrodes. The sensor is then
pushed all the way to the end of the housing and into the connector. This motion engages
the contact between the sensor contact trades and the connector springs.

The disclosed microprocessor based instrument is designed to perform various
tasks in the measurement of analytes, such as metal ions in biological and environmental
samples. The instrument advantageously operates as a stand-alone unit powered by a
battery or a AC power module, and is equipped with a communication port that allows
10 uploading analytical data to a computer. The unique electronic, microchip, and software
configurations developed for this device, have made possible a portable, low-cost, safe,
automated, and simple to operate instrument that is capable of precise and accurate
measurement of a wide range of analytes, including metals, peroxides, glucose, protons,
drugs, pesticides, eta

The innovative design of the disclosed apparatus incorporates a new data
processing method for attracting analytically useful signals from anodic sti^
In certain embodiments, the apparatus preferably employs a colloidal gold based electrode
that allows high sensitivity of detection of analytes so that exceptionally low levels of
20 analytes may be detectable.

An important feature of the present invention is the sensor lot calibration scheme.
The disclosed device may be set up for tests using different lots of sensors thereby
eliminating complicated and time consuming manual calibration strip procedures required
25 by other devices on the market attempting to perform similar analyses. There are two
general designs used in the devices for the calibration; one involves storage of specific
calibrations in the apparatus; another alternative and more versatile embodiment allows
calibration data specific for the sensor to be loaded into the apparatus prior to an analysis.

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The first version uses a calibration scheme that involves a resistor network-based
calibration strip and sensor calibration database stored in memory. It does not require any
user intervention except insertion of the calibration strip and pressing the START button.
The device recognizes that a calibration strip, and not a test strip, is connected and
5 conducts a measurement of resistance of two resistors on the strip. Based on the two
resistance values data from one of the calibration curves stored, the EEPROM is
transferred to the active portion of a lookup table. Until another calibration strip is read,
this data is used by the device to convert measured signals to the concentration of analyzed
species.

In a preferred embodiment, a novel version of a sensor lot calibration scheme is
employed that also allows setting up the operating parameters for a particular test in which
the sensor is used. It involves using a microchip, herein referred to as a "Calibration
Button,'' to store and download the calibration data and other data corresponding to a lot
IS of sensors that wQl be used in the test. A digital microchip reader of a size of a nickel coin
is mounted on the device that enables data transfer. Each lot of manufactured, sensors will
have a unique Calibration Button, and such a chip can beincluded with each set of sensors
sold from the lot

20 To prepare the memory chip, one may use a programmable memory chip such as

Dallas Senriconductor's Touch Memory. IK (64 data words), one time programmable
memory chips and programmable 4K memory chips are suitable. In one version of the
button, a 45-ppint calibration data set, a 7-point temperature correction data set, and the
sensor lot code and production date, in addition to the microchip ID code were stored.

25 Another version of the button sets up the device to a desired operating mode
(amperometric or square wave, for example) and changes the operating parameters,
including calibration, for the test and sensors to be used.

A memory reader probe, mounted on the enclosure, is used to transfer the
30 calibration data to the lookup table stored in the memory. When the device is in the

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READY mode, the Calibration Button reader is in the active mode and ready to sense the
attachment of the Button. The transfer is executed when the user connects (touches") the
button to the reader and the electromonitor device recognizes that a Calibration Button is
attached and ready for transfer of calibration data. Unlike the earlier described resistor
5 based calibration strip, the START key is not involved with the use of Calibration Biittoa
Transfer of data from the Button to the device takes less than 1 second.

Memory touch types of programmable chips are inexpensive and one calibration
button can be included with each set of sensors sold. Although the IK version is preferred,

10 the sensors may also be developed with a programmable 4K button. This allows storing
calibration data stored in the button's memory chip multiple times using a computer
program Programming of the buttons is fast; much less than 1 second per buttoa Buttons
may be purchased and no extra labor, except programming and labeling, is required For
certain applications, this presents an advantage over the calibration strip approach which

1 5 requires more elaborate and timely coordination of efforts between the sensor producer and
the monitor manufacturer.

The following material discusses the software and the hardware used in the
instrument. A brief overview describing the method utilized in the measurement is
20 presented first

Method Overview

The electronic device of the present invention measures an electrochemically
generated signal from an analyte in a drop of solution placed on a disposable sensor. The

25 instrument executes a sequence of voltage steps (voltage pulses) that are applied to the
electrodes on the sensor (FIG. 1). The electromonitor measures currents generated by the
sensor during the analysis. It then numerically processes these currents to determine the
analyte signal. In the final stage of the test, the electromonitor converts the analyte signal
to a corresponding analyte concentration in appropriate units and displays the result on an

30 LCD display.

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The electroanalytical techniques on which the operation of the dectromomtor is
based are square wave oculometry (SWC), anodic stripping vohammetry (ASV), and
amperometry. The SWC can be characterized as a hybrid of three electrochemical
techniques: Anodic stripping vohammetry, square wave vohammetiy, and oculometry. The
S dectromomtor can apply the appropriate analytical technique to measure signal for a
selected analyte. Because this measured signal is proportional to the concentration of
analyte in solution on the sensor, a simple conversion of this signal to the corresponding
analyte concentration can be performed using calibration data loaded in the memory of the
dectromonhor.

Hardware Overview

The block diagram of the system shows the basic hardware elements (FIG. 3). The
system in one embodiment may be built around a MOTOROLA MC6805 main
microprocessor 10, see also FIG. 8. The MC6805 is an 8 Wt microprocessor with 176
15 bytes of internal RAM, 8K bytes of program memory space, 24 I/O lines, 2 serial interfaces,
and a hardware timer. The 24 I/O lines and one serial port are used to connect to the
external components. The second serial port allows a host computer to communicate with
the system using a standard interface such as the RS-232 interface 12.

20 The analog circuit (FIG. 9) includes a digital-to-analog (D/A) converter 14, (FIG.

3), to generate a known voltage and an analog-to-digital (A/D) converter 16 to measure the
current. Additional Op Amps generate the counter dectrode voltage and measure the
reference voltage of the sensor and amvert the currem to a voltage for the A/D. Ananalog
switch allows disconnecting the dectronics from the sensor connector 18 when no sensor is

25 installed

The data collected while processing the sample is stored in an external 8K byte
RAM 20 (FIG. 10) for later analysis by the software. An EEPROM memory 22 may be
used to store the parameters for die measurement process. A lookup table, if incorporated
30 into the device, translates the result to the final displayed value in display 24.

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A power supply 26 for the system (FIG. 1 1) is provided either by internal batteries
or a 120V AC power module. A commercial 16 character display 24 may be used to
display messages and the final result of the measurement.

Software Overview

A support program for the unit allows a user to set the different parameters
associated with the process. These include timing and voltage levels for each state and
frequency of square wave modulation used in the scan state. In addition, the data may be
1 0 uploaded from the instrument and displayed.

A hardware prototype was built to provide a platform for the development of the
software. A MOTOROLA In-Circuft-Emulator was used to allow testing the code as it was
written. The software was broken into individually assembled small modules and then
15 linked together.

In addition, a simulation of the analysis routines was written in BASIC to allow
testing different methods for acquisition and analysis of data. This simulation in BASIC
was effective for development of one embodiment of the device, the LeadCare™ Monitor
20 used for the detection of lead in blood and was also used for optimization of the
LeadCare™ test system which includes special colloidal gold based electrodes used with an
dectromonhor calibrated and dedicated to lead testing.

Firmware Overview

25 The firmware can be divided into measurement of the data and communications to

the host computer. Used in the context of the present invention and as generally
understood by those skilled in the art, firmware refers to the software used as pan of the
disclosed device; that is, the software that is firmly fixed in the apparatus and which has
been especially developed for the embodiments disclosed and described herein.

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The measurement of data from the sensor is based on connecting the sample to a
voltage source for a fixed period of time and measuring the current. This system has been
developed as a general purpose tool; as a result, there is considerable flexibility in the device
for adapting to measure a wide range of types of analytes.

The firmware flowchart in FIG. 4 illustrates the steps in the process. Each step has
an associated time duration set by the user. This time can be set to zero, skipping that part
of the process. In addition, each step has a voltage applied to the sensor during that time.

10 The flowchart shows that the routine starts in a loop, waiting for the "START"

switch to be pressed. Once the switch is activated, each step is sequential. If the time is set
to zero, that step is skipped. The present system supports 4 stages: Initial delay,
Precondition #1, Precondition #2, and Deposition.

15 The next step is called the Scan stage (see Stripping Scan in FIG. 1). Thisstageis

more complex. The applied voltage is incremented from one level to a final level in a series
of steps. During each step, a small offset voltage is applied, first in the positive direction
then in the negative direction. During this positive offset time, the current in the ceil is
measured and stored as the FORWARD current (F). Similarly, during the negative oflfeet,

20 the current is measured and stored as the REVERSE current (R).

•' ■ .i
When the scan stage is completed^ the data analyas routine calculates the diflference

between these two currents and using this data, calculates the analyte level (FIG. 2).

25 The firmware was developed by creating a series of modules which handle one task

or function and then linking them together to form the total system. For the purpose of
illustration, the modules are grouped into three sections: software framework, data
collection and analysis, and support modules.

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PCT/US96/13844

Software Framework

The framework modules make up the program environment. This consists of a
main loop (FIG. 5) which polls and waits for signals to perform some activity. When a
signal is detected, it exits and runs those modules associated with that signal.

For example, timing is accomplished using hardware in the MPU to cause an
interrupt every 5 milliseconds. When this interrupt signal is detected, the firmware module
"TIMERIRQ" is run. This module handles the various time parameters such as the timing
for the stages during processing.

The framework modules consist of the "INIT" routine which initializes the
hardware and software memory, the main control loop in "MAIN" which tests for activities
ready to process, and the TIMERIRQ" routine which provides timing information.
Finally, the "VECTORS" module provides support for the MPU interrupts.

Data Collection and Analysis

The firmware which defines the function of the dectromonhor consists of seven
modules. The first module is in "MAIN." This module contains the basic testing loop. The
loop tests the status of the START switch, the status of the process if started, and if any
20 communication requests have been received from the host computer.

If the process has started, additional testing is done to support this mode. Tins
additional testing is primarily to determine if the time is complete for a stage and, if so,
setting up the next process stage. The timing is supplied by the basic framework routine
25 "TIMERIRQ" which generates a 1 -second signal which decrements the timer for the active

The last scanning stage is handled in the same way. The software for this stage,
however, isina separate module "SCAN" to allow easier testing and modification.

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Also, during the initial processing stages, the sensor is tested to determine if it is
dectrically working. This is accomplished by calling the routines in "STRIPTST" module.

The last four modules analyze the data collected and saved in the RAM buffer. The
"FILTER" module first smooths the data collected and calculates the difference values.
The "BASELINE" module corrects the data for the baseline ofiset. (See section on
analysis). Finally the result is translated into the correct BIX value using the routines in
"LOOKUP."

10 Support Modules

The remaining modules support the hardware, provide additional math routines and
test the system.

Hardware support routines:

LCD, DISPLAY:

RAM

EECODE:

ATOD, DTOA:

SERIAL, SERALIRQ:

BATTERY:

CALCSUM, CHECKSUM:
SELFTEST:
STRIPTST:
CALSTRIP:

hardware 16 character LCD unit
hardware external 2K memory
hardware EEPROM unit
hardware A/D,D/A
hardwareseriaIRS-232 interface
hardware,measurebatteryvoltage
hardware testing of program memory
hardware testing of analog circuits
hardware testing of sensor strip
test and measure calibration strip

Software support routines;

COMMANDS:

MATH:

ROUTINES:

DATA:

software math routines
software support routines

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PCT/US96/13844

The firnrware perfonns five internal test routines when started These routines
check the internal and external memory and analog hardware circuits.

The first routine tests the internal memory of the microprocessor unit. The internal
RAM memory used to store variables is checked and if an error is detected, the system
halts.

The second test checks the internal program memory or EPROM. This is done by
calculating the checksum of the internal memory and comparing it to a value previously
calculated and stored in memory. If the values are the same, the program memory is
acceptable; if not, the system halts.

The third set of tests checks the external memory. The first test checks the

■

EEPROM memory which contains parameters for the test Again a checksum is calculated
and compared with a value stored in memory. If the same, the memory is acceptable; if
different, then "SYSTEM ERROR" is displayed The lookup tables, if used in the device,
are also verified and if an error is detected, "SYSTEM ERROR" is displayed.

The external RAM buffer is also tested by writing a fixed pattern to the memory,
reading it bade and comparing it to the previously written data. If an error is detected, the
system displays "SYSTEM ERROR".

The final hardware test checks the D/A and AID circuits. This is accomplished by
outputting a known voltage from the D/A unit and measuring it using the AID unit. Three
vohages are output, -2 volts, 0 volts, and +2 volts. The result has to be within a preset
acceptable range. If an error is detected, the display shows "SYSTEM ERROR".

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PCI7US96/13844

Firmware Module Description

There are 31 firmware modules. A brief description of each is given in the
foDowing sections.

5 DEFINES.SRC

This module contains names of variables and their addresses in memory. The first
section contains the hardware locations with the corresponding names used in the software.
The remaining section contains variable names with their memory locations.

10 A 164)ft or 2 byte variable is label^

byte or low byte. A 24-bh or 3 variable is labded with "IT, TVT, *L" for high byte, middle
byte, and low byte.

ESTESCR

15 This routine initializes the system when power is first turned on. It is also entered

when the system has been powered down and the START switch is pressed to restart the
system. This routine performs the following tasks:

First, it initializes the hardware input/output ports, the serial peripheral interface
20 (SP*), the serial communications interfece (SO) and some internal registers.

It tests the internal RAM memory by first writing all ones to the memory location
followed by zeros. This leaves the memory reset to zero upon completioa

25 Next, the routine tests the internal program memory by doing a checksum total on

program memory space and comparing this to a prestored checksum value.

If either of these two tests fid, the system will not turn oa

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The hardware timer is set to generate interrupts every 5 milliseconds and the
interrupt logic is enabled.

The next section of code determines the source of the power for the instrument. If
5 the source is the DC power module input, no battery check is done. If the battery is the
source of power, the battery is checked and if low, a flag is set to display "BATTERY
LOW" message later. The power source check routine is repeated every minute during
normal operation of the unit.

10 The display is tested by turning on all segments and activating the beep for 2

seconds.

The calibration strip code is displayed for 2 seconds.

15 Finally, the display shows ,r READY." After the START switch has been released,

the system goes to the main entry point in MAIN.SRC.

MAIN.SRC

The MAIN.SRC section of code contains the primary system flow loop. This loop
20 consists of a series of questions that are sequenced through to determine which operation
should be performed. The loop is run every 5 milliseconds when the hardware timer causes
an interrupt and the software exits the "WATT 1 command.

The software first tests if the system is processing a sensor and is in the scanning
25 mode. If so' 9 it goes directly to the scanning software routine 30 in "SCAN.SRC" When
the routine is completed, h returns.

The next test determines if the process is active. If it is, the process control routine
is run (FIGS. 6A-6D). This routine is flow charted and will be discussed later.

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Next, the software determines if a command request has beat received from the
serial port If one has been received, it goes to the "COMMAND.SRC" routine 34 and
processes the command.

5 Finally, once every minute, the source of power is updated and the software loops

back to the WATT instruction 36 and waits for the next interrupt.

The process control routine (see FIGS. 6A-6D for start) controls the hardware that
is connected to the sensor during the test. The sensor test consists of a series of steps
10 which precondition the sensor electrodes by applying a voltage to the sensor for a given
period of time. The actual measurement scan is then initiated to collect the data which will
be later analyzed and an analyte value determined. There are 3 stages prior to the scan
routine: Initial Delay 40, Precondition #1 42 and Precondition #2 44.

15 Upon entry into this routine, the system determines if this is the first time. If this is

the case, it sets up the initial delay time. If the initial delay time is zero, then the system
jumps to the setup routine for Precondition # 1 . If the time is not zero it also checks to see
if the voltage is zero. If this is so, the analog switch does not connect the sensor to the
electronics. If the voltage is not zero, the analog switch is turned on and the software goes

20 to the exit routine.

Once the first setup routine has been run, die software loops to the time test The
remaining code is run every second. This is accomplished by monitoring a flag that is set by
the timer interrupt routine every second. If the flag is ofl£ the software goes to the test
25 command routine.

If the one second flag is on, the code first tests the electrical connections to the
sensor if the analog switch is oa Next it tests which stage is presently running and
continues that routine. These routines are all similar. First the time the stage is to be active

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is decremented and checked for zero. If the time is up, software goes to the setup routine
for the next stage. If the time is not up, it exits through the exit mode routine.

The setup routine starts by setting the new mode or stage number. Next it checks
5 the time delay for this stage and, if zero, skips and goes to the next stage.

The first part ofthe sensor test is next This test measures the current coming from
the sensor and saves the value. Next it outputs a new voltage for this stage. It remeasures
the current and compares it with that previously stored. There should be a difference due
10 to the new voltage applied. If not, an error is indicated and the error flag is set This error
flag will be handled in the exit mode routine.

The final stage sets up the scanning mode. It starts by displaying "PROCESSING."
Next it calculates a new voltage value based on the last voltage and step size and outputs
15 this to the sensor. Finally it updates the frequency counter and sets a flag to indicate that
the direction of current is positive, or forward. If the analog switch is not on, it is turned
on.

The exit mode routine occurs next It checks to see if any error was detected by
20 the two electrical tests ofthe sensor. If an error is detected, the display shows "STRIP
ERROR" and sounds a tone.

SCAN.SRC

The scan routine (FIG. 7) provides the actual data measuring software for the
25 sensor electrochemistry. This routine "scans" the voltage from the last voltage value up to
a final voltage in equal steps of 2mV increments. Given an initial voltage of -500 mV and a
final voltage of +50 mV, the software steps the voltage up in 275 steps of 2 mV.

WO 97/08544 PCT/US96/13844

During each step period, based on the frequency of the scan, an additional offset
voltage of first +25 mV and then -25 mV is applied and the resulting currents measured and
saved SeeFIG. 1.

The firmware to do this process consists of two routines, one for the positive offset
adjustment and one for the negative. When the positive offset is active, the current
measured is called the FORWARD current, and the negative offset current is called the
REVERSE cunent

1 0 The frequency of the scan is determined by the hardware interrupt timer rate which

is set at 5 milliseconds and the count in the frequency counter. For a frequency of 50 Hz
used the test the count is 2 which makes the period equal to 10 milliseconds or 20
milliseconds for the total square wave cycle.

15 The current is measured by a subroutine that actually measures the current four

times during each half of the square wave cycle and averages the results, This is done to
reduce effects caused by noise in the system. The calculation is done using 3 byte variables
due to scaling on the AID results.

20 The completed measurement is stored in the external RAM for later analysis. The

record format in the RAM is as follows:

N Forward current

N+l Reverse current

25 N+2 Voltage applied to sensor

N+3 Difference current (calculated at a latertime)

FBLTER.SRC

The filter routine performs four functions. It filters both the forward and reverse
current values in the external RAM buffers. Next it calculates the difference between

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fonvard and reverse currents and stores this in the buffer memory,
difference data before analysis.

The filter algorithm is a running average of 8 values. It is easy to divide by 8 simply
5 by shifting the result 3 times to the right. The eight values are chosen with' the value of
interest in the fourth position.

The routine starts by adjusting the starting and ending poirtere so vaHd data will be
used in the averaging process at the two ends of the data table. Next the table is scanned
1 0 adding up 8 values, dividing by 8 and storing the result

In calculating the difference, the code scans the memory buffer, subtracting the
reverse from the forward current value and storing the result in the last location of the data
record.

ANALYZE.SRC

The analysis routine is the most complex of the modules due to the number of math
operations. The routine to do the analysis was developed after encountering the finrited
capabilities of the microprocessor to perform complex calculations.

The data collected by the processor consist of the difference currents collected over
a voltage range defined by the parameters of the system. These difference currents, when
plotted yield a curve consisting of a peak superimposed on a baseline which is sloped (FIG.
2). The analysis routine first removes this sloped baseline and then calculates the area
25 under the peak portion of the curve. This area is the measured signal of analyte in the
sample.

The analysis routine works by noting that there is a minimum on either side of the
peak. Drawing a fine through the two minimum points and then subtracting the line from
30 the curve removes the baseline offset.

PCT/US96/13844

Finally it smooths

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The two minimum points are chosen by limiting the range that the software
searches. A low range is defined on the left side of the curve (at more negative voltages)
and a high range on the range on the right (at more positive voltages). Within these two
5 ranges, two minimum points are found and used to calculate the baseline.

The routine was first tested using a simulation written in BASIC from data
collected on samples. This showed that the routine worked best when run twice. Adjust
the data first, then run the routine a second time to further improve results.

The routine for running the analysis is set up in six steps:

1. Find the low point in the two ranges, the low and high

2. Calculate the slope of the line drawn between these two points
15 3. Subtract this baselines value from the data

4. Repeat 1 through 3 above to improve accuracy

5. Calculate the area between the two minimum points

6. Convert the area to a lead concentration value and display

20 The routine uses a series of subroutines to organize the above process and allow

testing of each step. The routine "CALCjvnN tt finds current value between two points
and is used to find the minimum for both the Ugh and low range.

Next the routines in "BASELINE" calculate the slope and the equation for the
25 baseline using the two minimum points. The software then subtracts the baseline value
from the difference current Then the "CALC_MIN n and BASELESS routines are repeated
to improve the accuracy of the signal measurement Finally, the last routine
"MEASAREA" calculates the area under the curve between the two mminnim points and
returns a numerical result in arbitrary (A/D) units.

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This value is then scaled into units of "peak area" so it can be compared with a
simulation program used to test the code. For example, in Mood lead measurements,
routine "LOOKUP" will take the signal value and convert it to a lead concentration result,
i.e.,aBLLinng/dL.

BASELINES.SRC

This routine removes the baseline offset from the original difference current curve.
The procedure is to calculate the equation for the best line fitting the curve at two points
and then subtract this line from the original data. The baseline equation is calculated by
knowing the two minimum points, e.g. 9 X2, Y2 which is the minimum point in the high
range and XI, Yl (the minimum point in the low range). Given these two points, the
equation for the line is:

Slope = ■

X2-XI

1) Calculate Y2-Y1, these are the difference current values. To increase the
overall accuracy, this value is scaled by 16.

2) Calculate the X2-X1 term, these are address values. The result is a number
of data points and is always positive.

3) Divide the above two numbers. This result is called the delta value and is
scaled by 16 to match the scaling of the difference current Values^

4) Next subtract the baseline from the difference current curve by starting with
the left minimum point. Subtract the minimum point value and then the delta value
multiplied by the position number. Continue this process until all the values of the
curve going to the right are calculated.

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5) In order to make the data look better when graphed, subtract the baseline
from the curve starting from the left minimum through zero.

LOOKURSRC

5 The LOOKUP module converts the area under the curve into a final analyte value

which is displayed. The routine uses a "lookup table" (see below) to perform the
conversion, demonstrated here for blood lead levels (BLL) in determining lead. Thetableis
organized into ten paired columns (EEPROM address paired with value). The table is
stored in the EEPROM memory by the program. The particular system described supports
10 eigjity eight (80) sets of calibration data The numbers are 16 bit words (2 bytes).

Column 1 of the lookup table (locations 000-0089) contains an active (working)
calibration curve. It is a set of 90 calibration values of the SWC signal, corresponding to
the BLL values in Column 2. This calibration curve is one of the 80 calibration curves
15 stored in Columns 3-10, and is used by the Monitor software to calculate the BLL from a
measured signal. The contort of this column is updated through an electronic calibration
process when a LeadCare Sensor calibration strip is used.

Column 2 (locations 0100-0189) contains a set of 90 BLL values covering the 8.5
20 to 62.5 jig/dL range in 0.6 pg/dL increments.

Column 3 through 10 (locations 0200-0299, 0300-0399, 0400-0499, 0500-0599,
0600-0699, 0700-0799, 0800-0899, arri 0900-0999) store right sets, eaA
values of the S^WC signal, representing eight different calibration patterns of the signal vs.
25 BLL dependence. Together with the BLL values in Column 1, the first 90 values of each
set represent a single calibration curve. The last ten values in each table represent 10
different ofl&ets for the upward adjustment of the stored calibration set.

Because of the limited number of entries in the table, the firmware interpolates
3 0 between two values to improve the resolution of the conversioa

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The lookup table contains information equivalent to 80 distinctively different
calibration curves. During the sensor calibration step, one of these curves is selected for the
measurement via a calibration strip supplied with each package of LeadCare Sensors. The
selected calibration curves are loaded into Column 1 (for example,
ARE A=ARE Al +offset 1 ) and then become working calibration curves in the measurement
ofBLL.

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L00KUP TABLES

EEPROM

Value

EEPROM

Value

EEPROM

Value

EEPROM

Value

EEPROM

Value

address

0000

AREA

0100

LEAD

0200

AREA1

0300

AREA2

0400

AREA3

0001

AREA

0101

LEAD

0201

AREA1

0301

AREA2

0401

AREA3

0002

AREA

0102

LEAD

0202

AREA1

0302

AREA2

0402

AREA3

0003

AREA

0103

LEAD

0203

AREA1

0303

AREA2

0403

AREA3

0004

AREA

0104

LEAD

0204

AREA1

0304

AREA2

0404

AREA3

0005

AREA

0105

LEAD

0205

AREA1

0305

AREA2

0405

AREA3

•

0089

•

AREA

0189

LEAD

0289

AREA1

0389

AREA2

0489

AREA3

0290

0FFSET1

0390

0FFSET2

0490

0FFSET3

0291

0FFSET1

0391

0FFSET2

0491

0FFSET3

0292

0FFSET1

0392

0FFSET2

0492

0FFSET3

0293

0FFSET1

•

0393

•

0FFSET2

0493

•

0FFSET3

•

0299

0FFSET1

•

0399

•

0FFSET2

•

0499

0FFSET3

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L00KUP TABLES (CONT'D.)

tcrnUM

value

CCDDflM

tcrnUM

value

CCPRflM
ctrnUIVI

Vali to

value

PFPRfl
ccrnu

1/ahtA
VolUc

FPPR
tern

Value

address

IVJ

urn

address

addre

0500

AREA4

0600

AREA5

ninn

0700

ADCAC

AncAb

nonn
UoUU

ADC A "7

AREA/

AAAA

0900

AREA8

0501

AREA4

0601

AREA5

A*tA4

0701

AREAS

0801

AREA7

0901

AREA8

0502

AREA4

0602

AREA5

0702

AREA6

AAAA

0802

AREA7

0902

AREA8

0503

AREA4

AAAA

0603 ,

. AREA5

0703

AREAo

0803

AREA7

0903

AREA8

0504

AREA4

0604

AREA5

0704

AREA6

0804

AREA7

0904

AREA8

0505
•

AREA4
•

0605

AREA5
•

0705

AREA6

■

0805

AREA7

•

0905

•

AREA8

•

0589

AREA4

0689

AREA5

0789

AREA6

0889

AREA7

•

0989

•

AREA8

0590

0FFSET4

0690

0FFSET5

0790

0FFSET6

0890

0FFSET7

0990 0FFSET8

0591

0FFSET4

0691

0FFSET5

0791

0FFSET6

0891

0FFSET7

0991' OFFSETS

0592

0FFSET4

0692

0FFSET5

0792

0FFSET6

0892

0FFSET7

0992

0FFSET8

0593

•

0FFSET4

•

0693

0FFSET5

•

0793

0FFSET6

•

0893

•

0FFSET7

0993

0FFSET8

0599

0FFSET4

0689

0FFSET5

0789

•

0FFSET6

0899

•

0FFSET7

0999 0FFSET8

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The firmware starts by testing that the "offset" value is not smaller or larger than
the minimum or maximum value in the first column (ofiset) of the table. If this is to be the
case, then the display will show "LOW 11 or "HIGH" correspondingly.

Next the first column of the table is scanned starting at the beginning for a value
that is larger than the "offset" value. When this is found, the previous position is saved as
"N n and the corresponding value in the lead column is saved.

10 As an example of using the Lookup tables in a typical analysis for blood lead levels,

the firmware will perform an interpolation calculation using the formula:

where ofiset ( ) are values in the offset column and LEAD ( ) are values in the LEAD
column, and offset is the original input value.

The final value is the LEAD(N) + Fraction. This is the final result of the BLL
measurement which is sent to the DISPLAY module for display.

STRBP.SRC

20 This routine contains two tests which are performed during the processing of the

sensor to determine if there are any problems with the sensor connections to the electronics.
The sensor has three connections: the reference electrode (REF), the counter electrode
(CE) and the working electrode (WE). A third test is needed to determine if the installed
sensor is a calibration strip.

The first test (STRIPTST) checks the electrical connection between the REF
electrode and the CE electrode. If the electrical connections are correct and the sensor

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electrodes are sufficiently covered by the test sample, the vohage of the CE electrode
should be the same as the REF electrode but of opposite polarity.

The firmware checks the CE and REF electrodes by measuring the voltage of each
5 and adding them together. The result should be zero or very dose to zero. The test checks
to confirm this difference is less than 100 millivolts. If the value is larger, an error flag is set
(the display shows CHECK STRIP).

This test is performed every second when the analog electronics is connected to the
10 sensor by the analog switch. (See MAIN. SRC)

The second test (TESTJWEl ,TESTWE2) checks the electrical connection to the
working electrode (WE). This test works by assuming that the current being measured by
the WE will change when the voltage being applied to the sensor by the CE changes.

The test is performed in two stages, the first (TEST_WE1) is to measure the
current before the voltage is changed saving this value. The second, (TEST_WE2),
measures the current after the voltage has changed and checks that it is different from the
first If the value has not changed by more than 125 nA the assumption is made that
20 something is wrong with the connection to the WE and the system signals an error (the
display shows CHECK STRIP).

The firmware actually checks to see if the voltage has changed by more than 50
millivolts due to limitations of the hardware. This should be noted in case the voltages from
25 one stage to the next do not change by more than tWsvahie.

This test is conducted twice, first when the voltage applied to the sensor is changed
during transition from PRECONDITION #1 to PRECONDITION #2 stage, and second
during transition from PRECONDITION #2 to DEPOSITION stage. This test is not run
30 when the analog switch is off It is also skipped if the time value for the stage is zero.

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The third test checks whether the sensor or a calibration strip is connected to the
device. The test is based on the cuiTent changing after the Precondition #1 voltage is
applied to a test sensor with sample. On the other hand, when a calibration strip is
5 connected the current does not change in time.

The routine first measures the current one millisecond into the Precondition #1 and
then every five milliseconds until two consecutive changes of more than 125 nA are
detected. When such changes are detected the routine assumes it is a test sensor. If the
1 0 current does not change within 2 seconds of the Precondition # 1 , a flag is set indicating that
the installed sensor is actually a calibration strip.

GALSTRIP.SRC

This routine determines the size of the two resistors on the calibration strip. This is
15 done by connecting each resistor to the output of the D/A converter, one at a time, and
sequencing the voltage up in steps of 10 mV until the output is 10 mA. This technique
allows using resistors in linear steps of 1 0 Kohms.

These two values are used to select one of the eight calibration tables stored in the
20 EEPROM. The second value is used to select one of the ten offset values in that table.
The offset value is added to all the values in the table to allow stuffing that data to best
match the characteristics of the LeadCare Sensor.

This is done by reading one value from the selected table, adding the selected
25 ofl&et, and storing the resulting value in the first table of the EEPROM.

The calibration strip code is displayed on the LCD when this process is completed .
The two-character code consists of a numeral (1, 2, 3, 4, 5, 6, 7, or 8) representing the
table number and a letter (A, B, C, D, E, F, G, H, I, or J) representing the offiet (A=l,

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B=2, etc). When the calibration strip is removed, the system goes bade to the "READY 1 '
mode.

As part of the sensor manufacturing process, each production batch of sensors is
5 calibrated. One of the 80 calibration curves which are stored on the device, is selected that
best matches the calibration data obtained for the current batch of sensors. This makes it
possible to assign a corresponding calibration strip which will be supplied with each
package of sensors produced in that batch.

10 CALBUmSRC
BUTNCMDS.SRS

These two modules support the touch memory system that is used to enter
calibration data into the monitor. The first module contains the two main routines, the first
detects if the touch memory is connected to the connector and the second reads the data
15 and transfers it to the EEPROM

The touch memory input system works by having the user touch the memory
button to the connector mounted on the outside of the housing. The first routine is called
every 10 miffisecbhds by the main polling routine to detect if the memory is connected.
20 When it is detected, the second routine is called which reads the data from the memory unit
and stores it in the RAM memory. The CRC value is checked and if correct, the data is
copied from the RAM to the EEPROM memory unit. If the data are not corrected, they
are read again and the test is repeated The system tries three times and then displays an
error message on the display and exits.

The additional module is used to support reading and writing the memory unit
using the MONITOR program. These routines allow the MONITOR program to write the
calibration data vahiesinto the touch memoryunit.

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UPDATE.SRC

This routine outputs data to the LCD display driver. The digital data are first
converted into segment data. The message symbols are obtained from flags in two
variables.

The data are shifted into the LCD driver using the SPI port and latched into the
output registers by toggling the chip select line.

DISPLAY.SRC

10 This module supports the LCD unit display which is a single fine, 16-character

LCD. The actual drivers for the display are in the module LCD. SRC. The data displayed
is based on the mode number in variable "LCDMODE. "

DISPLAY MODE:

1 Displays running information and time to complete process:
"TEST XXX sees" where XXX is the seconds remaining in the test

Displays result: "XXX" or "HIGH" or "LOW 1 in selected units

Displays "CALIBRATION ##" where ## is the code of calibration
strip

Routine "OJLLCD" clears the LCD unit except for the battery message symbol.

ATOD^SRC

This module supports a 12-bh analog to digital converter such as die LTC1296
(LINEAR TECHNOLOGY, Inc.). This A/D has 8 analog inputs and interfaces to the
microprocessor using the SPI, serial peripheral interface. The A/D is used to measure the
30 current from the test sensor, the battery voltage and additional internal voltages for testing.

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The firmware to support the A/D consists of two routines, one to read values and
one to power down the unit for power conservation

5 The first routine (AD READ) reads one input channel of the A/D and returns the

results in "ATODH, ATODL." This result is left justified in the 16 bit word. It is in 2s
complement notation. The voltage reference is 4.096 volts so the scale is 1 bit = 2
millivolts. The value is left justified in the 16 bit word.

1 0 The lookup table may be used following the routine to generate the address for the

input multiplexor.

The second routine (ADPWROFF) sends a command to the A/D converter that
puts it into a power down mode. In this mode the unit draws very little current. When a
15 conversion is requested, the unit powers backup.

DTOA.SRC

The DTOA module supports the MAXIM 12-bit digital to analog converter. The
microprocessor interfaces to the D/A using the SPI or serial pmphei^ imerfece. ThisD/A
20 has a built in 2.048 volt reference. An input value of zero gives the lowest vahie 6f-2X)48
wits. Thehigjhest 12^H vatoe of 4095 gives an oirt^

The formula for the output voltage is:

^ '

Output Volt =(-2.048) + N *(/miIlivolt)
where N is a number between 0 and 4095.

The firmware module outputs the value in variable "DAJiJDAJJ to the D/A
converter using the SPI port.

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RAM.SRC

This module supports the external memory which stores the collected data before it
is analyzed. The hardware implementation of the RAM consists of an 8K static RAM chip
5 and two 8-bit latches. The latches are loaded with the address of the data to be written or
read An 8-bit data bus, port C of the microprocessor, is used to load the two address
latches. The lower 8-bits of die address are output on the data bus and a control line
latches this data into the lower address. Sinrilaiiy, the upper 8-bits of data are output cm the
data bus and a second control line latches these data into the upper address .

This same data bus is also connected to the input/output port of the RAM. Once
the addresses are setup, another control line called READ/WRITE is used to set the RAM
mode. The CHIP SELECT control line causes the data to be read from the memory chip
or written into the memory chip.

For reading data from the memory chip, the data bus is changed into an input port
by setting the direction registers to zeros. When the port is an output port, the direction
registers are set to one. Also because the hardware address latches are not readable, a
software variable "RAMADRHJ," is used as the address data location. When a memory
20 operation is performed, these variables are used as the address information. All memory
operations are word or 2 byte operations.

The two firmware routines support reading the memory (RAMJRD) and writing
data to the memory (RAM_WR). Both routines first write the address to the latches and
25 read or write 2 bytes of data. Note that wlmcompleted, the address has been advanced to
the next word.

An additional routine allows advancing the memory address (ADVADR) by one

word.

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SERIALSRC

This module contains support routines for the serial communications to a host
computer. The serial hardware interface is setup to interrupt on incoming characters from
the host computer. The routine "SERIMJRQ.SRC" handles the interrupt and sets a flag
5 indicating a character is ready. The main routine polk this flag and if found set, goes to the
communication routines in "COMMANDS.SRC." In addition, if one of the commands
needs additional information, it calls routines in this module to get them

The first routine (INXHAR) gets one character from the host computer. It is

10 typically called by one of the command routines when requesting data from the host

computer. It polls the character ready flag and, when set, exits back to the calling routine

f ■
with the carry flag cleared It also has a time-out timer set to 1 second. If no character is

received in this time, h sounds a tone, sets the cany flag to indicate enor, and exits.

15 The next routine (INNUM) is used to input a number from the host computer.

The routine receives a character and tests if it is die end character (RETURN) and if so
exits with the number in "TEMPH^L." If h is a number, it is added to the previous
number by first multiplying the original number by 10 and then adding the new number. In
this way, any size number can be received.

Two routines are available to output characters or numbers to the host computer.
The firmware routine to output a character first tests if the RS-232 interface drip is
powered oa It is normally powered off to save power. Ifnot powered on, h is turned on
and a delay of 150 milliseconds allows the power to stabilize. The character is then output
25 to the host computer.

The second output routine is used to output a 2 byte value to the host computer as
a decimal number. First the value is converted to a decimal number, thai converted to
ASCII code and sent to the host computer.

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The last two routines send to the host computer either a single carriage return (CR)
or a combination of carriage return and line-feed (CR_LF).

SERALTRQ.SRC

5 This module supports the interrupt from the serial interface to the host computer.

The serial interface internal to the microprocessor is set to interrupt upon receiving a
character from the host computer; When this occurs, this routine is called. The firmware
receives the character from the serial port and tests for any errors. At this time, any
hardware errors are ignored and the software exits.

ThediaracteristestedtoseeiftfsaXONTROLC. If so, then the command
mode flag is reset, the analog switch is turned off and the cany flag is set to indicate error.
The purpose is to allow the external host computer to halt any command presently in
progress and cease any activity to the sensor in case the hardware should fail and lode up in
15 the command mode.

Next the character is tested for the START command, an "ESC" character. If it is,
the command flag is set so that the next time the main routine tests for a command
received, it will go to the command software routine.

Finally the character is stored in the receive variable and the data ready flag is set.

COMMANDS.SRC

The system was designed to interface to a host computer in order to allow the input
25 of new parameters for the process. In addition to these commands, additional commands
were implemented to allow testing the system during the development process.

A command packet from the host computer consists of an "ESC* character
followed by a single character which defines the command Some commands need an
30 additional number, this is entered as a decimal number ending with a carriage return.

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Below is a summary of the commands. A command can be halted by sending a
"Control C" character.

SYSTEM:

"R"

routine

Resets the system by jumping to the initialization

EEPROM:

"A" "N"

ii£ti njqit

RAM:

Loads the EEPROM address, from 0 to 1023
Writes 16-bit value into address
Reads 16-bit value from address

"M"

Loads the RAM address 0 - 2047
Writes 16-bit value into address
Reads 16-bit value from address

PROCESS:

"G"

Commands for testing:

Starts process

"B"
"IF

Sounds alarm tone

Runs filter routine on data in RAM buffer
Runs analysis routine on data in RAM buffer

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"L"

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Returns calculations of minimum points
Outputs value to D/A, #N = 0 to 4095
Returns AID value from channel #N = 0to7

ROUTTNES.SRC

The Pwr-Down routine is used to power down the system when it is turned off by
the software. It first turns off a transistor used as an on/off switch to all external hardware.
Next it sets up the microprocessor input/output ports so they can be powered down also.
Finally it puts the microprocessor into a "SLEEP" mode. The system will exit this mode
when the external "START switch is pressed causing an interrupt.

The BIN2BCD routine converts a 24-bit binary number stored in TEMPK^MJL
into five BCD numbers store in THOUSJ0, THOUS,HUNDRED, TENS, ONES. This is
done by subtracting first 10,000 from the binary number until the result is negative* thai
15 subtracting 1000, 100, and finally 10.

The BCD2BIN routine converts the BCD numbers in the five variables into a
binary number.

20 The DIVTOIO routine is used to divide the final result by 10 for display purposes.

It does this by first converting to a BCD value, shifting the BCD numbers by 1 position and
then reconverting to a Unary number.

The BEEPER routine drives the piezo transducer to create the sound referred to as
25 the "BEEP" or tone signal.

MATH.SRC

The math calculations contain two routines that perform mathematical functions.
The MULTI1Q routine multiplies the number in TEMPHML by It does this
30 using the formula:

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Multiplying by 2 and 8 are simple shift operations. The software generates the two
intermediate results and then adds them to get the answer.

5 The divide routine divides a 1 6 bit value in "TEMPM,TEMPL n by a 1 6-bh value in

W*N=2*N+8*N
"DIVSORItDIVSORL H and returns the results in "EMPMJEMPL."

EE_CODE.SRC

These two routines support reading and writing the EEPROM unit. This protocol
10 requires that to read the EEPROM the upper address and command mode data be sent,
thai a start sequence, the lower address data, and finally a stop sequence. Now the upper
address is resent, followed by a start sequence. Now the upper address is resent, followed
by a start sequence and the first byte of data can be read. The second byte is read next,
followed by a stop sequence sent to shut down the EEPROM.

The second routine (WRITE J3E) writes data into the EEPROM. Again the
EEPROM defines the protocol. First the upper address and stop sequence are sent Then
the lower address and the first byte to be written are sent, followed by the second byte to be
written and a stop sequence.

20 .

BATTERY.SRC

The battery test routine measures the battery voltage. Based on preset values, it
reports if the battery is OK, low or dead.

25 The battery voltage is divided in half by a resistor network before being connected

to the A/D converter. To compensate for the high resistance of the divider network, the
clock speed of the SPI port is reduced as is the clock to the A/D converter.

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The firmware starts by reading the battery voltage
measured twice and averaged to reduce noise in the circuits.

5 Next the voltage is compared to a preset minimum value that indicates that the

battery is OK If the voltage is above this amount, the error flags are cleared and the
system exits.

If the battery is below this preset minimum level, then the next step is to test to
10 determine if the battery is below the preset "dead" level Again, if the battery is above the
dead voltage level, the error flag for low battery is set and the system exits.

If the battery is below the operational voltage level, the analog circuits will not
work correctly. Even though the microprocessor system is functional, the system is hahed
15 by dearing the display and shutting off.

SELFTEST.SRC

This module contains four test routines which test the hardware when the system is
first turned oa An additional two tests are done in the JMT.SRC routine. These are the
20 tests of the internal RAM and the program memory of the microprocessor.

The first test (DATAJTST) checks the data stored in the EEPROMs first 20
locations which contain the parameters for the process run on the sensor. For example, the
EEPROM when loaded by the LeadCare Monitor program, stores in location 20 the
25 checksum of locations 1 through 19 inclusive of the EEPROM. This routine adds up these
locations (1-19) and compares the result with the value in location #20. If it is the same,
the data is correct If the two numbers are different, then the carry flag is set which causes
the display to show "SYSTEM ERROR #1" message.

The voltage is actually

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The second test checks the analog/digital converter and digital/analog converter. It
does this by outputting from the D/A a voltage and then measuring this voltage with the
A/D. This is not a perfect test because the reference voltage for both systems is the D/A
reference so, if it is not correct, the test may still work.

The test consists of three parts, first the D/A outputs a voltage of -2.0 volts,
measures this value and determines that it is within ± 2.5%. This error tolerance is to
compensate for small hardware offsets in the converters.^ The next test output is zero volts.
Finally the D/A outputs +2.0 volts. If an error is detected, the display shows "SYSTEM
10 ERROR#2."

The third test is to check the external RAM buffer memory. This is done with a
ample routine that writes the address of the location into the memory starting at location 0
and going to the end. It then reads the data and compares it with the address and checks
15 that they are the same. If an error is detected, this condition will cause the display to show
"SYSTEM ERROR #3."

At this time, the system error is reported but the system will still be allowed to run.
For a production unit, the system will be shut down upon detecting an error.

The fourth test checks the checksum of lookup Table #1 against the previously
calculated value stored in EEPROM. IF an error is detected, the display shows "SYSTEM
ERROR#4."

25 EXTTRQ.SRC

The external interrupt is generated whenever the START switch is pressed The
reason for having the switch connected to an external interrupt is to allow the system to be
restarted when it has been powered down and put into the sleep mode. If the switch is
pressed when powered down, the system is restarted by jumping to the initialization routine

30 INTT.SEC. IF the system is already powered on, the interrupt is ignored

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The START switch is also connected to an input port which is poDed in the
TIMERIRQ.SRC routine which monitors the switch and sets a flag if the switch is detected

OIL

TBVEERIRQ.SRC

The timer interrupt software is entered when the internal hardware timer of the
microprocessor causes an interrupt This interrupt rate is set for every 5 milliseconds and is
very accurate as it is based on a crystal timing circuit.

The timer works by having a free running 16-bit counter incremented by the
oscillator of the microprocessor. Internal circuits compare the counter value with another
167-bit register. When the two values are the same, it causes an interrupt to delay a fixed
time period, first read the present value of the hardware free running counter, add it to the
15 time delay and store this value in the compare latch. When the two latches compare, the
timeisup.

Upon entering this routine from an interrupt, the firmware restarts the timer
hardware. This is done by getting the present count value, adding to it the value equal to 5
20 milliseconds and storing this new value in the hardware compare register.

Next the counter decrements for the scan frequency. This wiD cause the scan
sequence to occur at the correct frequency rate. The remaining software is run every 10
milliseconds. A flag is checked to determine if this is the second interrupt and if so, the
25 code continues, otherwise it exits.

The remaining code is run every 10 milliseconds. It first tests the condition of the
START switch. This routine debounces the switch by requiring that it be stable for 10
milliseconds before a flag is set indicating that it is on.

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The next routines are used to decrement the timers that are based on the 10
millisecond time. These are used in the rest of the code to delay actions or act as time out
timers.

5 The next section of code is activated only every second and is used to update timers

that are based on 1 second time intervals.

Finally the last section of code is based on 1 -minute intervals and is used to
decrement the time the unit is active. If this time becomes zero, then the software goes to
10 the routine that powers off the system Note that the power down timer is reset whenever
the START switch is pressed or a character is sent from the host computer.

CALCSUMLSRC

This routine is used during the final production of the software to calculate the
15 checksum of the program memory space. It is run using the ICE development system
When run and halted, the CHECKSUM value is in the accumulator This value is then
stored in the checksum data field in the module DATA. SRC .

When the system is first turned on, a routine is run that calculates the program
20 space checksum This value is compared with the value stored in the data space. If it is the
same, the program space is accepted If different, the program memory space is no longer
correct and the system halts.

CHECKSU1VLSRC

25 This routine is ran when the system is first turned on or powered back on by the

START switch: This routine adds up all of program memory space to generate the
checksum value. This value is compared with a value stored in the data memory location.
If the two values are comparable, the program space is accepted. If the values do not
agree, the program space is not correct and the system halts.

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The checksum in the data memory space is calculated by the CALCStMSRC

routine.
DATA^RC

5 This module contains data which is stored in the program memory space. The first

location contains the checksum of the program space for testing. The remaining space is
available for storing information about a product.

VECTORS.SEC

10 This module is linked into the program space at the veiy top and contains the

vector addresses for the different interrupts.

The following examples illustrate particular applications of the herein disclosed
electrochemical analyzer. It will be readily apparent to a skilled artisan that changes,
IS modifications and alterations may be made to the disclosed apparatus and software
combined therewith without departing from the true scope or spirit of the invention.

EXAMPLE 1

20 The disclosed device is conveniently used to detect lead in water. FIG. 14 is a

calibration curve for lead in water using anodic stripping signals measured by the monitor
with a colloidal gold sensor. The electrochemical monitor parameters were: 90s deposition
at -0.5V and stripping by square wave vohammetry at 1 00 Hz, 25 mV amplitude and 2 mV
steps. The medium was 0.125 M HGL

25.

FIGS. 17A-17B compare anodic stripping curves as acquired and after processing
bytheinventioa The curves were obtained using a 42 fig/dL lead in add treated blood and
measured using a colloidal gold sensor. Operating parameters included a 90s deposition at
-0. 5 V and stripping by square wave vohammetry at 80 Hz frequency, 25 mV amplitude and
30 2 mV steps.

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The device was also used to test for lead in the presence of various metals. Results
are shown in Table 3.

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TABLE3

Solution tested

+0ppbPb

+12.5ppbPb

+125ppbPb

As,ppb

Cr(VI),ppb

Hgipb

Se,ppb

run#1

run #2

run#1

run #2

run#1

run #2

120 dilution
ofTCLP
Concentrate
#1

1000

0.1

0.6

0.5

44.8

48.5

1:2000
dilution of
TCLP

Concentrate
#1

100

5.1

8.1

85.2

TO.2

without
TCLP

Concentrate
#1 added

6.2

8.4

106.1

106.0

Solution tested

+50ppbPb

Ba,ppb

Cr(VI),ppb

Ag,ppb

Cd, ppt

Pb,ppb

run#1

run #2

1:10,000 dilution of TCLP

Results showed that the calibration curve in the absence of tested interferences showed
good linearity and reproducibility in the range of 0-100 ppb Pb. Reproducibility of the
SWC signal at 12 ppb Pb level was good with a SD of 1.8 for 12 measurements with an
average value of 8.7. The test solution containing As, Cr(VT),Hg and Se ions (TCLP
10 solution) reduced the Pb signal when present at a 100-fold excess over Pb. At a 10-fold
excess, there was no interference with this solution. TCLP solution #2 contained Ba, Cd,
Cr(VI), and Ag and was found to increase the Pb signal at 1 00-fold excess over Pb.

As illustrated in FIG. 15, an anodic stripping curve was obtained for a mixture of
Zn, Cd. Pb and Cu employing a carbon sensor with an m situ deposited mercury film

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bstrument parameters were: 240s deposition at -1.4 V and stripping by square wave
vohammetry at 1 15 Hz frequency, 25mV amplitude and 3 mV steps.

EXAMPLE 2

The disclosed device was also used to determine cadhim concentrations. Using a
colloidal gold sensor strip, samples containing cadhim ion were placed on the sensor and
analyzed using the following instrumental parameters:

1) Delay Time And Voltage 0 Sec 0 mV

2) Precondition #1 Time And Voltage 10 Sec 500mV

3) Precondition #2 Time And Voltage 10 Sec 50mv

4) Set Deposition Time And Voltage 90 Sec 500mv

5) Set Equilibration Time 0 Sec

6) Set Voltage -Final 200mv

7) Set Step Voltage, # Of Steps 2rav 351 Steps

1) Delay Time And Voltage
9) Set Gain, Freq

G) Start Test And Retrieve Data (D)

R) Review Data

A) Analyze Data

S) Save Data

L) toad Lookup Table

OSec
0-10 jxA

OmV
80HZ

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The calibration curve for cadium ion is shown :

Cd (ppb)

Table 4 shows the SWC signal obtained for different amounts of Cd in the sample.

TABLE 4

Cd, ppb

Signal

5.3

125

250

116

Experiments involving copper(II) and cadmium(II) proved that both metals were
dectroactive on colloidal gold modified graphite ink electrodes and could be cathodically
deposited on and anodically stripped off the gold surface. Under the solution and
instrumental conditions used for measurement of lead, copper and cadmium produce
IS stripping signals that are proportional to their respective concentrations in the ppb range.
The data (not shown) indicated that copper, cadmium and lead can each be determined in
the absence of the remaining two.

WO 97/08544

PCT/US96/13844

-57-

Simultaneous determination of more than one heavy metal in water containing
several metal ions is readily accomplished with the disclosed system. One can selectively
shift peak potentials of stripping peaks by complexing the metal ion, which can be
accomplished by manipulation of pH and addition of specific ligands. Thus a test for
5 simultaneous determination of the three metals from a single stripping scan is possible.

An alternative approach to determining a heavy metal in the presence of other
heavy metals would be to selectively mask to allow determination of individual metals in the
presence of other electroactive metals. For example, one can eliminate the anodic stripping
10 peak of copper by adding EDTA and lowering pH of the sample to 1.0. In contrast to
copper^, lead(H) is not bound to EDTA at this pH and can be measured without
interference from copper. Similar approaches using other ligands and buffering systems are
expected to allow masking of other metals to determine an analyte metal in waters
containing mixtures of metals.

EXAMPLE 3

FIG. 13 shows the use of the electromonitor for the determination of a drug. The
figure shows a square wave vokammetric curve of acetaminophen employing a carbon
20 sensor. The parameters used were: square wave vokammetry with 100 mV initial
potential, 50 Hz frequency, 25 mV amplitude and 2 mV steps.

FIG. 16 illustrates the use of a colloidal gold-HRP (horseradish peroxidase) sensor
to measure hydrogen peroxide in 50 mM MES buffer, pH 6.4 using the disclosed device for
25 amperometric measurement. Parameters were: -100 mV potential, 4 Hz sampling rate.

EXAMPLE 4

A temperature change may severely affect current signals using sensor and
30 arrangements that do not protect the sample solution from evaporatioa As shown in

WO 97/08544 ... PCT/US96/13844

-5S-

FIG. 1 8, solution evaporation caused by low humidity produces a rapid drop in temperature
on the sensor. Lower temperature results in decreased diffusion coefficient of the analyte
to the sensor's working electrode which can reduce the measured signal This effect can be
reduced by using a heat sink platform enclosed in the sensor housing.

Further modifications and alternative embodiments of this invention will be
apparent to those skilled in the art in view of this description Accordingly, this description
is to be construed as illustrative only and is for the purpose of teaching those skilled in the
art the manner of carrying out the invention It is to be understood that the forms of the

10 invention herein shown and described are to be taken as the presently preferred
embodiments. Various changes may be made in the software and firmware; for example,
the calibration data may be provided in multiple diskettes, partially provided from external
data bases, or stored in the device itself for later analysis. Multiple ports for sensor insert
may be provided and adaptations for analysis of several different analytes may be

15 incorporated Certain features of the invention may be utilized independently of the use of
other features, all as would be apparent to one skilled in the art after having the benefit of
this description of the inventioa

WO 97/08544

PCT/US96/13844

-59-
CLAIMS :

1. Apparatus for measuring the amount of an analyte in a liquid electrolyte which
comprises:

a housing substantially enclosing a data processing system including a power
supply, a microprocessor, a memory and an electrical connector,

a sensor positioned connected to the housing and comprising a working electrode,
a reference electrode and a counter electrode adapted to be in contact with
a sample of said electrolyte and to be connected with said electrical
connector so as to couple the signal output of the sensor with the data
processing system;

a digital data memory containing calibration data interrelating values of said signal
output with amounts of said analyte in said electrolyte;

said microprocessor programmed to apply a potential to said sensor, to measure the
signal output from said sensor, and to compare said measured signal output
with said calibration data to thereby determine the amount of said analyte in
said electrolyte.

2. An apparatus as defined in claim 1 in which said digital data memory comprises a
calibration strip positioned outside the housing and adapted to be connected with said
electrical connector.

3. An apparatus as defined in claim 1 in which said digital data memory comprises a
look up table.

WO 97/08544

PCT/US96/13844

-60-

4. An apparatus as defined in claim 1 in which said digital data memory comprises a
microchip positioned outside the housing, and said apparatus further comprises a microchip

5 reader operable to down load calibration data from said microchip to said data processing
system.

5. An apparatus as defined in claim 1 which further comprises a structural module
1 0 attached to said housing and configured to screen said sensor from drafts.

6. An apparatus as defined in claim S wherein said structural module includes a metal
plate extending from said housing to support said sensor and serve as a heat sink for said

15 sensor.

7. Apparatus for use with a sensor to analyze for a metal analyte in an aqueous
electrolyte, wherein the sensor comprises at one end a reference electrode, a counter

20 electrode, and a working electrode which has a colloidal gold surface, said apparatus
comprising:

a housing adapted to be hand held and to support said sensor with said electrodes
for contact with said electrolyte;

a microprocessor controlled source of potential in said housing operable to apply
said potential to said working electrode sufficient to deposit said analyte
relative on the working electrode;

WO 97/08544

PCT/US96/13844

-61-

a microprocessor controlled source of providing a sequence of potential steps
operable to apply said steps over a range of potentials sufficient to strip said
analyte from the working electrode wherein the microprocessor operates to
control the potential to step said potential upward from said lower end of
5 said range in steps of equal potential and duration so as to strip the analyte

from said working electrode thereby generating a current and reverse
current;

said microprocessor also operable to measure and process said forward and reverse
10 current to generate a signal and to compare said signal with signals similarly

obtained from known quantities of said analyte in said electrolyte so as to
thereby determine the unknown quantity of said analyte in said electrolyte.

15 8. The apparatus as defined in claim 7, wherein the analyte is lead and the electrolyte
comprises blood or urine.

9. The apparatus as defined in claim 8 wherein said lower end is about -500 millivolts,
20 the upper end of the range is about +100 millivolts, said square wave alternates about ± 25
millivolts, and each step is about 3 millivolts.

10 An apparatus for analyzing for a metal analyte in an aqueous electrolyte,
25 comprising:

a housing adapted to be hand-held;

a sensor positioned outside the housing and adapted at one end to be mechanically
30 coupled to the housing and at another end to be contacted with said

WO 97/08544 PCT/US96/13844

-62-

electrolyte, said another end including a reference electrode, a counter
electrode and a colloidal gold working electrode; and,

a microprocessor in the housing programmed to:

a) apply a sequence of potential steps to the working electrode ova* a
potential range from an initial level to a final level sufficient to strip an
analyte deposited on said working electrode;

10 b) measure the forward and reverse current generated by said sequence of

c) process to obtain a charge signal to measure the total amount of net current
from said forward current and said backward current generated between

15 said first level and said second level; and,

d) compare said signals with signals obtained from calibration of said
apparatus using known amounts of said metal analyte in said aqueous
electrolyte under the same operating conditions as for the unknown

20 quantity, and thereby determine the quantity of said analyte in said

electrolyte.

11. The apparatus of claim 10 in which the microprocessor is further programmed to
25 apply at least one constant potential to said working electrode for a selected period of time
to deposit an analyte on said working electrode prior to actuating said sequence of potential

WO 97/08544 PCT/US96/13844

-63-

12. The apparatus of claim 11 in which said metal analyte is lead, said initial level is
about -500 millivolts, said final level is about +100 millivolts, said alternating voltage is
about ± 25 millivolts and in the form of a square wave, and each said step is about 3
millivolts.

13. The apparatus of claim 11 which further comprises a lookup table containing said
reference totals.

14. The apparatus of claim 11 which further comprises a display for displaying the
analyzed quantity of said analyte.

15 15. Apparatus for use with a sensor to analyze for a metal analyte in an aqueous
electrolyte, wherein the sensor comprises at one end a reference electrode, a counter
electrode, and a working electrode which has a colloidal gold surface, said apparatus
comprising:

20 a housing adapted to be hand held and to support said sensor exterior of the

housing with said electrodes in contact with said electrolyte;

a microprocessor controlled source of potential in said housing operable to apply
said potential to said working electrode sufficient to deposit said analyte
25 relative on the working electrode;

a microprocessor controlled source of providing a sequence of potential steps
operable to apply said steps over a range of potentials sufficient to strip said
analyte from the working electrode wherein the microprocessor operates to
30 control the potential to step said potential upward from said lower end of

WO 97/08544

PCT/US96/13844

-64-

said range in steps of equal potential and duration so as to strip the analyte
from said working electrode thereby generating a current and reverse
anient;

5 said microprocessor also operable to measure and process said forward and reverse

current to generate a signal and to compare said signal with signals similarly
obtained from reference values similarly obtained from known quantities of
said analyte in said electrolyte so as to thereby determine the unknown
quantity of said analyte in said electrolyte.

16. The apparatus as defined in claim 15, wherein the analyte is lead and the electrolyte
comprises blood or urine.

17. The apparatus as defined in claim 16 wherein said lower end is about -500
millivolts, the upper end of the range is about +100 millivolts, said square wave alternates
about ±25 millivolts, and each step is about 2 millivolts.

18. A portable system for measuring the amount of an analyte in an aqueous electrolyte
comprising:

a housing enclosing a microprocessor controlled source of potential, a digital data
25 processing system and a microprocessor based data processing system;

an electrochemical sensor positioned outside said housing, said sensor having a
working electrode, a reference electrode and a counter electrode at one end
to contact said aqueous electrolyte and adapted at its other end to transmit
3 0 signals generated by said electrolyte to said data processing system;

WO 97/08544

-65-

PCI7US96/13844

said source of potential adapted to apply potentials across said sensor sufficient to
cause said sensor to generate and transmit current to said data processing
system through said housing;

a source of calibration data positioned outside said housing correlating current
values generated and transmitted by said sensor for different amounts of
said analyte in said aqueous electrolyte and at said potentials;

10 said microprocessor programmed to compare currents generated and transmitted

by said sensor in response to said potentials with said calibration data to
thereby ascertain the amount of said analyte in said aqueous electrolyte.

IS 19. A system as defined by claim 1 which further comprises a visual display positioned
within said housing, and circuitry to display the amounts of analyte ascertained on said
display.

20 20. A system as defined by claim 1 wherein said source of calibration data comprises a
microchip encoded with such data, and wherein said system further comprises a microchip
reader mounted on said housing and adapted to download calibration data from said
microchip to said digital data processing system.

21. A system as defined by claim 1 wherein said calibration data comprises is selected
by a calibration strip.

WO 97/08544 PCT/US96/13844

-66-

22. A microchip encoding the values of signals generated by an electrochemical sensor
in contact with a given electrolyte containing given amounts of an analyte and at given
potentials or a sequence of potential steps applied to the sensor, said sensor including a
reference electrode, a working electrode and a counter electrode.

23. A microchip as defined in claim 22 wherein the working electrode comprises
colloidal gold. _

24. The apparatus of claim 1 wherein the potential is a single potential step.

25. The apparatus of claim 1 wherein the potential is a sequence of potential steps.

26. The apparatus of claim 1 wherein the sensor is positioned outside the housing.

20 27. The apparatus of claim 1 wherein the sensor is positioned inside the housing.

WO 97/08544

PCT/US96/13844

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WO 97/08544

PCT/US96/13844

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INTERNATIONAL SEARCH REPORT

International / 1 cation No

PCT/US 96/13844

A. CLASSIFICATION OF SUBJECT MATTER

IPC 6 G01N27/42 G01N33/487

According to International Patent Qasaficaboo (IPC) or to both national classification and IPC

B. FIELDS SEARCHED

Minimum documentation searched (classification system followed by classification symbols)

IPC 6 GQ1N C12Q

Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched

Electronic data base consulted during the international search (name of data base and, where practical, search terms used)

C. DOCUMENTS CONSIDERED TO BE RELEVANT

Category ' Citation of document, with indication, where appropriate, of the relevant passages

Relevant to daim No.

A,P

US 5 468 366 A (WEGNER STEVEN ET AL) 21

November 1995

cited in the application

see the whole document

US 5 437 772 A (DE CASTRO EMORY S ET AL)
1 August 1995

see column 7, line 10 - column 10, line
32; figures 1.3A

US 5 366 609 A (WHITE BRADLEY E ET AL) 22
November 1994

see column 1, line 7 - column 4, line 55

1-27

1,2,7,8,
10.

14-16,
18-22,
24-27

1.2,

18-22,

24-27

Further documents are listed in the continuation of box C

Patent family members are listed in annex.

• Special categories of cited documents :

"A* document defining the general state of the art which is not

considered to be of particular relevance
*E* earlier document but p ublishe d on or after the international

filing date

"L* document which may throw doubts on priority daixn(s) or
which is cited to establish the publication date of another
citation or other special reason (as specified)

"O* document referring to an oral disdosure, use, exhibition or

T later document published after the international filing date
or priority date and not in conflict with the application but
cited to understand the principle or theory underlying the

"P* document published prior to the international filing date but
later than the priority date claimed

"X* document of particular relevance; the claimed invention
cannot be considered novel or cannot be considered to
involve an inventive step when the document is taken alone

*Y* document of particular relevance; the daimed invention
cannot be considered to involve an inventive step when the
document is combined with one or more other such docu-
ments, such combination bring obvious to a person skilled
in the art.

'ft* document member of the saroc patent family

Date of the actual completion of the raternational search

21 January 1997

Date of mailing of the

- 3. 02. 97

Name and mailing address of the ISA

European Patent Office, P.B. 5815 Patenttaan 2
NL - 2280 HV Rijswi*
TeL (+31-70) 340-2040, TX. 31 651 epo nL
Fax (+31-70) 340-3016

Authorized officer

Brrson, 0

Form PCTTS A/210 (wnrf I*** IW)

page 1 of 2

INTERNATIONAL SEARCH REPORT

International / i cat) on No

PCT/US 96/13844

^Continuation) DOCUMENTS CONSIDERED TO BE RELEVANT

Category* Citation of document, with indication, where appropriate, of the relevant passages

Relevant to claim No.

US 5 217 594 A (MENKENS ROBERT W ET AL) 8
June 1993

cited in the application
see abstract; figure 7

US 5 120 421 A (GLASS ROBERT S ET AL) 9
June 1992

see column 4, line 61 - column 15, line 65

EP 0 653 629 A (EC0SSENS0RS LIMITED) 17
Hay 1995

see column 4, line 37 - line 40

see column 8, line 1 - line 40; figure 6

EP 0 351 516 A (P0RT0N INTERNATIONAL INC.)
24 January 1990
' see column 11, line 35 - column 12, line 9

7,8,10,
14-16

7,10,15,
18

7,16,15,
18

Form PCT/BA/Iltl |aa»utt>» of «OM tt»M) (JulT tWT) .

page 2 of 2

INTERNATIONAL SEARCH REPORT

Inform^ ja on patent family members

International / 'i cation No

PCT/US 96/13844

Patent document

Publication

Patent family

Publication

cited in search report

dale

mem

ber(x)

due

US-A-5468366

21-11-95

US-A-

5368707

29-11-94

US-A-

5217594

98-06-93

AU-A-

3734095

26-64-96

WO-A-

9610741

AU-A-

3474193

US-A-

5334296

W0-A-

9314185

t-C V/ ?3

US-A-

5225064

06-07-93

US-A-5437772

61-08-95

NONE

US-A-5366609

22-11-94

AU-A-

7093694

93-01-95

CA-A-

2153884

22-12-94

EP-A-

0746762

11-12-96

JP-T-

8502590

19-03-96

WO-A-

9429703

22-12-94

US-A-5217594 08-06-93

AU-A-

3474193

03-08-93

US-A-

5368707

29-11-94

US-A-

5334296

02-08-94

WO-A-

9314185

22-07-93

US-A-

5468366

21-11-95

US-A-

5225064

06-07-93

US-A-5120421 09-06-92 US-A- 5296125 22-03-94

EP-A-0653629 17-05-95

AT-T-

126888

15-09-95

AU-A-

6880091

26-06-91

OE-D-

69021888

28-09-95

DE-T-

69021888

11-04-96

EP-A-

0504196

23-09-92

ES-T-

2077213

16-11-95

WO-A-

9108474

13-86-91

US-A-

5512489

30-04-96

EP-A-351516 24-01-90

AU-A-

1529692

09-07-92

AU-B-

625758

16-07-92

AU-A-

3507789

18-01-90

CA-A-

1316572

20-04-93

JP-A-

2147050

06-06-90

Form PCT/TSA/210 (poUat fimtfy annex) (July 1992)

page 1 of 2

INTERNATIONAL SEARCH REPORT

Infcrnw. on patent family member*

Intemanonat .' 'tcaboo No

PCT/US 96/13844

Patent document
cited in search report

Publication

Patent family
inemberfs)

Publication
date

EP-A-351516

US-A-

5998545

24-63-92

Form PCTflSAJHO (pHtxA tern* *bmx) <J«ty 1W3)

page 2 of 2

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